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Transcriptional regulation of mouse mu-opioid receptor gene
J L Ko1, H C Liu, S R Minnerath
1Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Abstract:
Previously, the existence of dual promoters was reported in mouse mu-opioid receptor (mor) gene, with mor transcription in the mouse brain predominantly initiated by the proximal promoter. In this study, we further analyzed the proximal promoter region, base pairs -450 to -249, to identify cis-DNA regulatory elements and trans-acting protein factors that are important for mor promoter activity. The results revealed that a mor inverted GA (iGA) motif and a canonical Sp1 binding site are required for the promoter activity. Using electrophoretic mobility shift analysis, we identified nuclear proteins that specifically bind to the mor iGA motif and that are immunologically related to Sp1 and Sp3. Mutation of the mor iGA motif, resulting in a loss of Sp binding, led to a 50% decrease in activity. Mutation of the canonical Sp1 binding site yielded a lesser (approximately 25%) loss of activity. Mutation of both motifs together resulted in an approximately 70% decrease in activity. In cotransfection assays using Drosophila SL2 cells, Sp1 trans-activated the promoter in a manner dependent on the presence of mor iGA and canonical Sp1 binding motifs. Sp3 can also trans-activate the promoter, and furthermore, Sp1 and Sp3 can trans-activate the mor promoter additively. Our results suggest that combined or cooperative interaction of Sp transcription factors within the proximal promoter is necessary for activation of mor gene transcription.
Insights
This study identifies key DNA elements and Sp transcription factors regulating mouse mu-opioid receptor (MOR) gene expression. Cooperative binding of Sp1 and Sp3 to the MOR proximal promoter is crucial for gene activation.
Area of Science:
- Molecular Biology
- Neuroscience
- Genetics
Background:
- The mouse mu-opioid receptor (MOR) gene possesses dual promoters, with the proximal promoter primarily driving transcription in the brain.
- Understanding the regulatory mechanisms of the MOR proximal promoter is essential for comprehending MOR gene expression.
Purpose of the Study:
- To identify cis-DNA regulatory elements and trans-acting protein factors governing MOR proximal promoter activity.
- To elucidate the roles of specific motifs and transcription factors in MOR gene regulation.
Main Methods:
- Analysis of the MOR proximal promoter region (-450 to -249 bp).
- Electrophoretic mobility shift assays (EMSA) to identify protein-DNA interactions.
- Site-directed mutagenesis of identified motifs.
- Cotransfection assays in Drosophila SL2 cells to assess promoter activity and transcription factor function.
Main Results:
- A MOR inverted GA (iGA) motif and a canonical Sp1 binding site are critical for MOR promoter activity.
- Nuclear proteins immunologically related to Sp1 and Sp3 bind to the MOR iGA motif.
- Mutation of the iGA motif decreased promoter activity by 50%, while mutation of the Sp1 site reduced it by 25%; combined mutations caused a ~70% decrease.
- Sp1 and Sp3 trans-activated the MOR promoter, with additive effects observed when both factors were present.
Conclusions:
- The MOR proximal promoter activity is dependent on the presence of both the iGA motif and the canonical Sp1 binding site.
- Sp1 and Sp3 transcription factors play a significant role in activating MOR gene transcription.
- Cooperative interactions among Sp transcription factors within the proximal promoter are necessary for efficient MOR gene expression.